1. Field of the Invention
The present disclosure relates to a screening method of therapeutic agents for essential tremor by using a mouse model in which essential tremor (or hand tremor) is enhanced.
2. Description of the Related Art
Essential tremor is a typical disease, showing action tremor or postural tremor. Action tremor or postural tremor refers to a type of tremor aggravated throughout active movement, or during posturing such as “holding a cup” or “stretching the arms forward”. On the contrary, a tremor, which worsens when motionless and at rest, refers to resting tremor, and often occurs in Parkinson's disease. Essential tremor is a hereditary disease inherited through autosomal dominant inheritance, and thus tends to have high incidence among the family members of an essential tremor patient. Thus, essential tremor is also called familial tremor. The diagnostic characteristic of essential tremor is that it occurs slightly less frequently but more severely in most cases than physiological tremor during normal physiological activities. However, the tremor can not be easily distinguished from other forms of tremors only by apparent symptoms. As a patient ages, tremors tend to increase in amplitude and decrease in frequency. Tremor may be evident only in the upper extremities, and may occur in the head. When tremor is serious, it may occur in the jaw, lips, tongue, and even in the vocal cords, and tremor in the latter causes the voice to tremble when speaking. Even though essential tremor causes relatively mild symptoms, it varies with each individual and begins with minute tremor in one side or in both sides of the body and progresses slowly. These symptoms occur when there is a change in posture or moving state and are not detected in a stable phase unless the disorder is in an advanced stage. No other neurological disorders related to systemic or neuronal disease are caused. It is easy to diagnose this disorder due to its familial tendency, and tremors can sometimes be temporarily alleviated by consumption of alcohol.
Methods for treating essential tremor are based on the use of drugs. Conventional methods that have been used include drinking alcohol or the intake of an alcohol compound (octanol), and taking inhibitory drugs such as a receptor antagonist of the inhibitory neurotransmitter GABA. Inhibitory drugs such as Primidone or Propranolol as a beta-blocker are primarily used. These drugs exhibit about 60% to about 70% efficacy on patients, but are ineffective at completely eradicating the symptoms. When the efficacy does not satisfy expectations, treatment may introduce a secondary therapeutic agent or a physical operation. However, the receptor antagonist of GABA or alcohol compound can interfere with normal functions of the nervous system in addition to tremor, and moreover causes serious side-effects including sleep induction, etc. Dangerous brain surgeries such as thalamectomy or deep-brain stimulation should only be performed as a last resort. While deep-brain stimulation has recently received increasing attention as a relatively safe and reliably effective method for treating tremor, it is quite costly.
The gamma-aminobutyric acid type A (GABAA) receptor superfamily represents one of the classes of receptors through which the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) acts. Widely, although unequally, distributed throughout the mammalian brain, GABA mediates many of its actions through interaction with the GABAA receptor, which causes alteration in chloride conductance and membrane polarization. In addition to the neurotransmitter such as GABA, a number of drugs, including the anxiolytic and sedating benzodiazepines, also bind to the receptor. In general, the GABAA receptor includes a chloride channel that opens in response to GABA, allowing chloride ions to enter the cell. This, in turn, effects a slowing of neuronal activity through hyperpolarization of the cell membrane potential.
GABAA receptors includes five protein subunits, typically composed of two α subunits, two β subunits and one γ subunit. Each subunit includes α 1-6, β 1-3, and γ 1-3 (Mohler, H. et al, Neuroch. Res, 20, 631-636, 1995). The α 1 subunit of the conventional GABAA receptor was subjected to a knockout procedure to develop a mouse model (GABAA receptor α 1−/− mouse; Vicini, S. et al, The Journal of Neuroscience, 21, 3009-3016, 2001) exhibiting essential tremor genetically at 17-22 Hz (Kralic, J. E. et al, The Journal of Clinical Investigation, 115, 774-779, 2005). However, there are limitations in these studies on essential tremor and development of therapeutic agents because the tremor intensities were too weak at an early age.
Voltage-dependent calcium channels play a role in increasing the concentration of calcium in a cell by the activity of neural cells (Tsien, R. W., Annu Rev Physiol 45, 341-358, 1983), and are classified into high-voltage dependent and low-voltage dependent channels according to the voltage dependency (Tsien, R. W. et al, Trends Neurosci, 18, 52-54, 1995). T-type calcium channels are an exemplary low-voltage dependent calcium channel, and there are three kinds of Cav3.1 (α1G), 3.2 (α1H), and 3.3 (α1I) in mammals according to the genotype of α1 subunit. α1G calcium channels are involved in production of burst firings of nerve cells in the thalamic nucleus, and their major pathological functions have been recently revealed (Kim, D. et al., Science, 302, 117-119, 2003; Kim, D. et al., Neuron, 31, 35-45, 2001). The present inventors conventionally disclosed that α1G T-type calcium channel-deficient mice have resistance against essential tremor induced by harmaline, a tremor-inducing agent.
Thus, the present inventors have constructed a double knockout mouse (α1−/− /CaV3.1−/−) by mating a GABAA receptor α1−/− mouse which exhibited essential tremor symptoms with an α1G T-type calcium channel knockout mouse which had resistance against essential tremor, studied the essential tremor symptoms of the double knockout mice, observed that the tremor intensity of the double knockout mouse was more severe, unlike that of the conventional GABAA receptor α1−/− mouse which failed to meet the expectation that the essential tremor would be improved, enough to be visually confirmed even at an early age, and completed the present invention by confirming that the mouse model may be used as a model for development of therapeutic agents for essential tremor.
The present inventors also have constructed an α1F/+; Emx1-Cre mouse by mating a GABAA receptor α1F/F mouse with an Emx1-Cre and constructed an α1−/−; Emx1-Cre by mating the α1F/+/Emx1-Cre mice with each other. The inventors studied the essential tremor symptoms of the conditional knockout mice, observed that the tremor intensity of the α1−/−; Emx1-Cre mouse was more severe than that of the conventional GABAA receptor α1−/− mouse, and completed the present invention by confirming that the mouse model may be used for the study of essential tremor, for the screening of a drug for essential tremor and as a model for the study of dorsal telencephalon (cortical) functions related to essential tremor.